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Bacillus cereus capable of producing cellulase and xylanase at low temperature

A Bacillus cereus, cellulase technology, applied in the field of microbial engineering, can solve the problems of low enzyme production enzyme activity, little research on Bacillus cereus enzyme production, discounted enzyme activity effect, etc., and achieves high enzyme activity, excellent pH resistance. The effect of acceptability and good enzyme activity

Active Publication Date: 2021-07-23
JILIN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There are a variety of microorganisms in the natural environment, but most of them are greatly reduced due to the limitation of ambient temperature or bacterial growth rate. Usually, the temperature is low, the growth of microorganisms will be inhibited, and the enzyme activity is low. Especially in the cold environment in the north for a long time, there are few studies on the production of enzymes for the survival of Bacillus

Method used

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  • Bacillus cereus capable of producing cellulase and xylanase at low temperature
  • Bacillus cereus capable of producing cellulase and xylanase at low temperature
  • Bacillus cereus capable of producing cellulase and xylanase at low temperature

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Screening and identification of the original bacterial strain TK-1

[0022] (1) Isolation of strains

[0023] (1) Collect 5g of soil samples in the forest area of ​​Changbai Mountain Nature Reserve, dissolve them in 100mL of prepared saline solution, shake and cultivate at 10°C and 180rpm for 2-4h, and the saline solution is 0.006mM FeSO 4 ·7H 2 O; 0.01 mM CaCO 3 ·7H 2 O; 0.08mM MgSO 4 ·7H 2 O; 0.07mM MnSO 4 ·7H 2 O and 0.006mM ZnSO 4 ·7H 2 O composition.

[0024] (2) Dilute 1mL of the mixed soil sample salt solution with different gradients (10-2, 10-3, 10-4), take 200ul of the diluted solution and spread it on the R2a solid plate, and place it in a constant temperature culture at 10°C Cultivate statically in the box for 7-10 days. The fixed plate contains 0.5g yeast extract powder, 0.5g peptone, 0.5g casein hydrolyzate, 0.5g glucose, 0.5g soluble starch, 0.3g potassium dihydrogen phosphate, 0.024g anhydrous Magnesium sulfate, 0.3g sodium pyruv...

Embodiment 2

[0041] Embodiment 2: original bacterial strain TK-1 mutagenesis treatment

[0042] Using ARTP mutagenesis, the mutagenesis steps are as follows:

[0043]The power is 100W, the mutagenic working gas is helium, the gas flow rate is 12s / m, and the distance is 3mm to irradiate for mutagenesis. After diluting the mutagenized bacterial suspension, take 100 μL of the diluted solution and spread it on a PDA plate. Cultured in an incubator. Take 1 mL of the bacterial solution cultured to the logarithmic phase (OD600 between 0.6 and 0.8) in a 1.5-ml centrifuge tube, centrifuge at 10,000 rpm for 2 minutes, discard the supernatant, and resuspend the bacteria twice with normal saline to obtain a cell suspension. Dilute to adjust cell number at 10 6 -10 8 pieces / ml.

[0044] Take 10uL of the cell suspension and evenly spread it on the surface of the metal slide (sterilized), and adjust the parameters of the ARTP instrument:

[0045] The treated slides were transferred to a centrifuge t...

Embodiment 3

[0047] Embodiment 3: the growth and enzyme production analysis of Bacillus cereus TK-2

[0048] (1) Analysis of growth characteristics of Bacillus cereus TK-1 and TK-2

[0049] (1) The bacterial strain TK1 and the mutant strain TK-2 that grow well at low temperature and produce cellulase and xylanase obtained by mutagenesis are respectively inoculated in LB liquid medium, at a rotating speed of 180rpm, different temperatures (10 , 15, 20, 25, 37°C) for shaking culture, sampling at fixed points, and measuring the OD of the bacterial solution 600 value.

[0050] (2) Analysis of enzyme production characteristics of Bacillus cereus TK-1 and TK-2

[0051] (1) Inoculate the logarithmic phase bacterial liquid in the fermentation enzyme production medium (ammonium sulfate 4g / l, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.1g / L, yeast extract 10g / L, peptone 1g / L, xylan / CMC-Na 1.5%), the inoculum volume was 200uL, the rotation speed was 180rpm, and shaking ...

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Abstract

Bacillus cereus capable of producing cellulase and xylanase at low temperature is preserved in the China Center for Type Culture Collection. The preservation address is No. 299, Bayi road, Wuchang District, Wuhan City, Hubei Province; the classification name is Bacillus cereus TK-2; and the preservation number is CCTCC NO: M2021433, and the preservation date is April 22, 2021. The bacillus cereus TK-2 has low-temperature tolerance and adapts to growth and enzyme production at a relatively low temperature; particularly, the enzyme activity of xylan produced at a low temperature of 15 DEG C is 2.3 times that of xylan produced at a temperature of 37 DEG C; the enzyme activity of produced xylanase can reach 60.57 IU / mL; the enzyme activity of cellulase (endo-cellulase) is 28.2 IU / mL; and the produced xylanase and cellulase still have high enzyme activity at a relatively low catalytic temperature, have excellent pH tolerance, and have relatively good enzyme activity in weak acid, neutral and alkaline environments.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to a bacillus cereus producing cellulase and xylanase at low temperature. Background technique [0002] Xylan (xylan) is a five-carbon polysaccharide, the main component of hemicellulose in plant cell walls, and another abundant regenerative polysaccharide in nature besides cellulose. Xylanase (xylanase) can convert xylan into xylooligosaccharides and xylose, and has broad application prospects in agriculture, energy, feed, papermaking, food and other fields. There are many sources of xylanase, among which the xylanase produced by microorganisms is one of the main sources. At present, due to the limited variety of xylanase-producing strains, high production costs, and insufficient enzyme-producing ability of strains, the industrial application of xylanase is restricted. Therefore, it is of great significance to develop new strains of xylanase with high activity and w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12N9/24C12R1/085
CPCC12N9/2437C12N9/248C12Y302/01004Y02E50/10
Inventor 张斯童沈丽君包昌杰陈欢王刚孙旸陈光
Owner JILIN AGRICULTURAL UNIV
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